Neuropeptide FF (NPFF) is the mammalian homolog in the family of peptides related to the molluscan peptide FMRFamide. NPFF has been implicated as an anti-opiate peptide in morphine analgesia, and in the development of tolerance, dependence. and withdrawal. A binding assay for NPFF receptors was developed in membrane preparations of rat brain and spinal cord. The NPFF analog 125I-YLFQPQRFa bound specifically with 0.08 nM affinity to a single population of sites in spinal cord membranes. NPFF receptors were found to be regulated by sodium and magnesium ions and GTP. We found that NPFF receptor binding was highest in brain regions high in NPFF, and the levels of binding in saline-treated and morphine-dependent rats were not different. The NPFF analog daY8Ra reduced morphine tolerance in rats. These results may have implications in treatment of drug addiction and chronic pain. The proposed course of this project includes (1) study of SAR of peptide binding to NPFF receptors, (2) investigation of the coupling of NPFF receptors to cellular signal transduction systems, (3) development of specific NPFF antagonists, and (4) solubilization and purification of NPFF and FMRFamide receptors.